A Resource for Oxygen Imaging by Phosphorescence Quenching Project Summary Real-time, minimally invasive and spatially resolved measurements of tissue oxygenation have the potential to transform our understanding of many clinical problems, including those in tumor biology, management of stroke, ophthalmology, tissue regeneration, to name a few. Over the years our laboratory has developed a minimally invasive method for dynamic imaging of oxygen in biological systems, known as phosphorescence quenching oximetry. Recently, the method has been expanded by two-photon phosphorescence lifetime microscopy (2PLM), enabling dynamic imaging of oxygen gradients in tissues in 3D with micron-scale resolution, opening new horizons for research in neuroscience, stem cell biology, cancer immunology, tissue engineering and several other areas. Even more recently, the combination of phosphorescence with Cherenkov-Excited Luminescence Scanned Imaging (CELSI) enabled tomography of oxygen in 3D in pre-clinical setting in tissues undergoing radiation therapy. At the same time, phosphorescence-based oximetry is increasingly drawing attention from the medical community for its potential to directly evaluate physiologic status of tissue under trauma, optimize efficacy of blood transfusion as well as a marker in monitoring progress of photodynamic and radiation therapies. These and other applications have set the stage for broad dissemination of the phosphorescence technology across different biomedical fields. At the heart of the phosphorescence quenching method are special oxygen probes, developed and continuously optimized in our laboratory. The synthesis of these probes, including the newest and the most potent probe Oxyphor 2P, is not simple, and standard commercialization pathways are presently not feasible. Here we propose to establish a U24 Resource that would allow us to sustain synthesis of phosphorescent probes, making them available to a broad range of biomedical researchers across different fields. Simultaneously, we will generate software for measuring/imaging oxygen by phosphorescence lifetime and will establish a center to provide consultations and training of new users interested in the method. The work will be performed at two closely collaborating sites: the University of Pennsylvania (probe chemistry, software development) and Martinos Center for Biomedical Imaging at the MGH (software development, user training). Our laboratories have long history of productive collaboration as well as multiple collaborations and contacts with researchers interested in oxygen. These collaborations along with the past experience of running a neuroscience research resource will help us to establish an effective program, making the phosphorescence- based oximetry accessible to a broad user base.